Attenuated T cell responses to a high-potency ligand in vivo

Abstract

αβ T cell receptor (TCR) recognition of foreign peptides bound to major histocompatibility complex (pMHC) molecules on the surface of antigen presenting cells is a key event in the initiation of adaptive cellular immunity. In vitro, high-affinity binding and/or long-lived interactions between TCRs and pMHC correlate with high-potency T cell activation. However, less is known about the influence of TCR/pMHC interaction parameters on T cell responses in vivo. We studied the influence of TCR/pMHC binding characteristics on in vivo T cell immunity by tracking CD4(+) T cell activation, effector, and memory responses to immunization with peptides exhibiting a range of TCR/pMHC half-lives and in vitro T cell activation potencies. Contrary to predictions from in vitro studies, we found that optimal in vivo T cell responses occur to ligands with intermediate TCR/pMHC half-lives. The diminished in vivo responses we observed to the ligand exhibiting the longest TCR/pMHC half-life were associated with attenuation of intracellular signaling, expansion, and function over a broad range of time points. Our results reveal a level of control over T cell activation in vivo not recapitulated in in vitro assays and highlight the importance of considering in vivo efficacy of TCR ligands as part of vaccine design.

Figure 1. Kinetic parameters of the interaction of the 5C.C7 TCR with four peptide/I-E^k ligands.

(A) Surface plasmon resonance at 25°C was used to study the interaction of varying concentrations of soluble 5C.C7 TCR with the indicated immobilized peptide/I-E^k complexes as described in Materials and Methods. The concentration of soluble TCR resulting in each RU trace is indicated on the MCC/I-E^k plot, and applies to the corresponding traces on the K3/I-E^k and K5/I-E^k plots. (B,C) Comparison of all four peptide/I-E^k complexes is shown at 7.1 µM and 14.3 µM TCR, respectively. Association (k_a) and dissociation (k_d) constants were calculated as described in Materials and Methods and are shown in Table 1. (D) Dissociation of PE-labeled H2-I-E^k tetramers bearing the indicated peptides from naïve 5C.C7 RAG2-deficient T cells was done at 25°C or 37°C. TCR/pMHC tetramer half-lives were calculated from linear regressions and are shown at the end of each line and in Table 1.

Figure 2. K5 peptide is a more potent inducer of in vitro 5C.C7 T cell proliferation than MCC peptide when presented on both in vitro and in vivo pulsed APCs.

(A) Lymph node cells from 5C.C7 TCR transgenic RAG2−/− mice were stimulated with peptide-pulsed irradiated B10.A splenocytes in the presence of ³H-methyl-thymidine for 60 h. The mean EC₅₀ for each peptide derived from these experiments is shown in Table 1. B10.A mice, three per peptide group, with (B) or without (C) adoptively transferred 5C.C7 RAG2−/− T cells, were immunized with the indicated peptides and LPS, and 2 d later CD11c+ splenocytes were purified, irradiated, and used to stimulate naive 5C.C7 RAG2−/− T cells in vitro for 60 h. (D) Titration of exogenous MCC peptide added to the in vivo–pulsed APCs in (C) (10⁵ per well) shows that the APCs are equally capable of presenting antigen. Data in Figure 2 are representative of at least three independent experiments. Error bars show mean ± s.e.m., n = 3 wells.

Figure 3. Immunization with MCC peptide and LPS results in expansion, contraction, and maintenance of 5C.C7 T cells in vivo.

(A) B10.A mice with adoptively transferred naïve 5C.C7 RAG2−/− CD45.1 T cells were immunized with LPS alone (“no peptide”) or LPS+MCC peptide (“MCC”). CD4⁺CD45.1⁺ lymphocytes were detected in blood (“PBL”) and lymph node (“LN”) samples 6 d after immunization (LN CD4⁺ were purified by negative selection before flow cytometry). (B) Time course of 5C.C7 expansion, contraction, and maintenance in blood in response to immunization with MCC peptide. The traces of four individual mice are shown.

Figure 4. Blunted in vivo responses of 5C.C7 T cells to a high-affinity ligand.

Naïve 5C.C7 RAG2−/− CD45.1 T cells were adoptively transferred into normal B10.A recipients and activated by immunization with the indicated peptides and LPS. (A) Day 2.5 CFSE plots represent samples pooled from four or five mice, and number of divisions is indicated at the top of each peak. Frequency of 5C.C7 T cells as a percentage of total CD4⁺ T cells, or frequency of 5C.C7 T cells producing IL-2 or IFN-γ, are shown in samples from day 6 lymph nodes on representative flow plots. (B) Proliferative capacity was calculated from CFSE profiles, and 5C.C7 T cells were quantified as a percentage of total CD4⁺ T cells. Because of the low frequency of 5C.C7 T cells present at day 2.5 it was necessary to combine samples from multiple mice to visualize CFSE profiles and analyze proliferative capacity. Thus, each bar in the graphs represents samples pooled from four or five mice, and the data are representative of three independent experiments. (C) Upper left, number of 5C.C7 T cells (normalized to total CD4⁺ T cells) in day 6 lymph nodes; the data are pooled from four independent experiments. Horizontal lines on graphs represent the mean. ***p<0.0001. Upper right and lower left, number of IL-2⁺ and IFN-γ⁺ cells (as a percentage of 5C.C7 T cells). IL-2, *p = 0.0104. IFN-γ, *p = 0.0463. The lower right panel shows the absolute number of IFN-γ⁺ 5C.C7 T cells per 1×10⁷ lymph node CD4⁺ T cells. **p = 0.0078. Cytokine data are pooled from two independent experiments. Two-tailed p values are from unpaired t test.

Figure 5. Fewer K5-stimulated 5C.C7 T cells are found at the peak, contraction, and maintenance phases of the response.

5C.C7 T cells were adoptively transferred and activated as in Figure 3, and blood samples were analyzed for CD4⁺ CD45.1⁺ cells at the indicated time points (A). Number of 5C.C7 T cells expressed as a percentage of total CD4⁺ T cells is shown. Error bars show mean ± s.d. Day 6, *p = 0.0247, n = 5. Day 90, *p = 0.0257, n = 5. Data are representative of two independent time courses. (B) shows CD62L staining of day 59 5C.C7 T cells from lymph nodes. Number of 5C.C7 T cells in lymph nodes is shown at day 31 (C, ***p<0.0001) and day 59 (D, ***p<0.0001). Two-tailed p values are from unpaired t test. Data in (B), (C), and (D) are representative of two or more independent experiments.

Figure 6. In vivo activation status of 5C.C7 T cells stimulated with the high-affinity ligand K5 peptide.

Naïve 5C.C7 RAG2−/− CD45.1 T cells were adoptively transferred and activated by immunization with the indicated peptides and LPS. (A) Lymph node CD4⁺ T cells were stained for PD-1 at day 2.5 and day 6 (CD4⁺CD45.1⁺ gated). The day 2.5 histograms represent samples pooled from four or five mice. The black histogram corresponds to cells stimulated with LPS alone. Graphed PD-1 MFIs are from day 6, error bars show mean ± s.d., n = 3 mice. (B) Annexin V staining on CD4⁺ T cells from day 6 lymph nodes. (C) Day 2.5 lymph node samples were fixed immediately after harvest, methanol permeabilized, and stained with antibodies to phosphorylated Akt (S473) and phosphorylated Stat3 (Y705). Histograms are gated on CD4⁺CD45.1⁺ cells and represent samples pooled from four or five mice. Percent of 5C.C7 T cells positive for annexin V, pAkt, or pStat is shown on the plots and histograms. The data are representative of three independent experiments.